Comparative transcriptomics of stem rust resistance in wheat NILs mediated by Sr24 rust resistance gene

Stem rust of wheat is a deleterious fungal disease across the globe causing severe yield losses. Although, many stem rust resistance genes (Sr) are being used in wheat breeding programs, new emerging stem rust pathotypes are a challenge to important Sr genes. In recent years, multiple studies on leaf and yellow rust molecular mechanism have been done, however, for stem rust such studies are lacking. Current study investigated stem rust induced response in the susceptible wheat genotype C306 and its Near Isogenic Line (NIL) for Sr24 gene, HW2004, using microarray analysis to understand the transcriptomic differences at different stages of infection. Results showed that HW2004 has higher basal levels of several important genes involved in pathogen detection, defence, and display early activation of multiple defence mechanisms. Further Gene Ontology (GO) and pathway analysis identified important genes responsible for pathogen detection, downstream signalling cascades and transcription factors (TFs) involved in activation and mediation of defence responses. Results suggest that generation of Reactive Oxygen Species (ROS), cytoskeletal rearrangement, activation of multiple hydrolases, and lipid metabolism mediated biosynthesis of certain secondary metabolites are collectively involved in Sr24-mediated defence in HW2004, in response to stem rust infection. Novel and unannotated, but highly responsive genes were also identified, which may also contribute towards resistance phenotype. Furthermore, certain DEGs also mapped close to the Sr24-linked marker on Thinopyrum elongatum translocated fragment on wheat 3E chromosome, which advocate further investigations for better insights of the Sr24-mediated stem rust resistance.


Introduction
Wheat is the third most important cereal crop globally, and crucial for economic as well as nutritional security world-wide [1].Wheat production is affected by numerous biotic (rusts, smuts, powdery mildew and fusarium head blight) and abiotic (heat, drought, shorter season duration, salt and alkalinity) stresses [2].Among the biotic stresses, rusts of wheat are important threat to global wheat production [3].Due to rapidly evolving virulent pathotypes and large area of impact, rust diseases have become a major challenge for wheat breeders [4].Stem rust of wheat a highly deleterious disease (caused by Puccinia graminis f. sp.tritici (Pgt)), is estimated to cause up to 50% loss in yield or even higher if infection starts at an early stage [3].Effective management of stem and other rusts involve employment of resistance genes (R) and chemical intervention [2].Close to 60 Sr loci are now reported, including race specific all duration resistance genes as well as broad-spectrum adult plant resistance (APR) genes [5].Although chemical intervention is still utilized to control wheat rusts, genetic control of the disease using 'R' genes is still the most economical and environment sustainable approach [3,6].
Resistance mediated by 'R' genes is based on its successful interaction with corresponding Avirulence (Avr) factors of pathogen (incompatible reaction).Failure to recognize the Avr factors lead to susceptibility and disease development (compatible reaction) [7].Plant defence mechanisms against pathogens is based on immune responses operating at two-levels [5,8].The primary/basal defence mechanism directed against a large number of pathogens involves PRR-mediated recognition of conserved PAMP, and is termed as PAMP Triggered Immunity (PTI) [9].Here, the transmembrane PRR protein having LRR (extracellular) and kinase (intracellular) domain binds to pathogen-specific signatures, activating defence mechanisms orchestrated by PR proteins, ROS modulation and cell wall strengthening [9,10].Plant pathogens can evade the detection by the PRRs by secreting the effector molecules directly inside the host cell, using specialized secretion system [8].However, plants use 'R' gene to detect these effector molecules and activate second level of defence response, termed as Effector Triggered Immunity (ETI), which typically results in hypersensitivity response (HR) [11,12].The 'R' proteins are classified into five groups, of which the NBS-LRR (NB-ARC-LRR) group is the largest [13].In ETI interaction between effector and 'R' gene receptors may be direct, or it may detect the modifications on the host cell surface, leading to a relatively rapid defence response than PTI [11,14].
Multiple 'R' genes have been cloned, however, the mechanisms responsible for conferring resistance are not understood completely [15].Reports on wheat leaf and yellow rust have provided insights into important candidate genes and pathways involved in resistance mediated by both race-specific and APR genes, particularly on, resistance mechanisms in mixed races (field inoculum) [16][17][18][19].Emergence of new virulence Pgt pathotypes (e.g.Ug99 group), has resulted in breakdown of several important 'Sr' genes viz.Sr31 and Sr24, which is alarming for breeders and pathologists [3][4][5].Hence, detailed studies are needed for understanding molecular basis of resistance conferred by different 'R' genes against stem rust disease in wheat.Unlike leaf and yellow rust, global analysis of resistance response is lacking in case of wheat stem rust.Sr24 (linked to Lr24, chromosome: 3D), an important Sr gene present in global wheat varieties, confers resistance to most of stem rust races (includes Ug99 race TTKSK, all races in India except 62G29-1) [20][21][22].Being originated from alien source Thinopyrum elongatum, its molecular understanding is not well known, however, due to presence of tightly linked DNA markers (e.g.Xbarc71, Sr24#12) it has been widely deployed in many wheat varieties across the globe [22,23].The wild relative of wheat T. elongatum, (referred to as tall wheat grass, E genome) has been utilized for transferring multiple resistance genes to wheat, including Sr24 [23,24].Gene transfer using wide hybridization from T. elongatum resulted in translocation stock lines at 3D, 3DL, 3BL and 1BS [25][26][27][28].Further, the 3D/Ag lines developed by Sear's were used for developing white seed wheat varieties in Australia and was then used as source for breeding Indian varieties [29].
In this study, NILs for 'Sr24' were used for understanding transcriptomic differences between resistance and susceptibility upon challenge with a local stem rust race 7G11.Infection with 7G11, lead to highly susceptible reaction on recurrent parent C306 and resistant type reaction on HW2004.High-throughput microarray analysis revealed peak transcriptional difference at early stages of infection, with upregulation of genes for pathogen detection and defence mechanism activation.Overall, genes for receptors, activation of signalling cascade, TFs for defence genes, and defence responses including ROS, secondary metabolites, and PR proteins showed upregulation in resistant NIL (HW2004) at early stage of infection.Certain Differentially Expressed Genes (DEGs) were also mapped on to the T. elongatum translocated fragment, and in vicinity to the Sr24-linked marker, including both the uncharacterized as well as disease responsive genes.

Plant material and rust inoculation
Wheat stem rust susceptible variety C306 (lacking Sr24 gene) and its NIL genotype HW2004 with Sr24 (also known as Unnath C306; C306 + Sr24), were obtained from Indian Agricultural Research Institute Delhi.The plants were grown in an MLR-351H plant growth chamber (SANYO, Japan) under following conditions: 16 hours light/ 8 hours dark cycle, temperature: 25˚C (light) 18˚C (dark) and humidity: 80-95%.Puccinia graminis f. sp.tritici (Pgt) pathotype 7G11 spores were received from IIWBR Regional Station, Flowerdale, Shimla, (infection type 1 on HW2004, 4 on C306) these were multiplied and maintained on C306.Wheat seedlings (at GS13, Zadok scale) were inoculated with stem rust spore suspension (concentration: ~6x10 5 spores ml -1 in water containing 1 ppm Tween 20 detergent) as described by Bhardwaj 2011 [30,31].Stem rust infection phenotyping was done after 14 days post inoculation (dpi).Leaf tissue samples were collected at three time-points i.e., 0h, 10h and 72h post inoculation (hpi), snap freezed in liquid nitrogen, and stored at -80˚C till further use.For each time-point tissue samples of three plants were pooled together, and experiment was repeated after one month for the second independent biological replicate.

Microarray analysis
Total RNA was extracted from leaf tissue (500 mg) using TRIzol (Life Technologies, USA) method, and treated with DNase I enzyme (Invitrogen, USA) as per manufacturer's instructions.Quantification of RNA preparations was done on Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific, USA) and integrity was assessed on 2100 Bioanalyzer (Agilent Technologies, USA).Good quality RNA samples (OD 260/280 values: 1.8-2.2,28S/18S rRNA ratio: 2:1, and RNA integrity number (RIN) of >7) were used for microarray analysis, carried out at Genotypic Technologies Pvt Ltd. (Bangalore, India).Total RNA was labelled using Agilent Quick Amp labelling kit (part number: 5190-0442) as per the recommended protocol.Briefly, total RNA (500 ng) was reverse transcribed using oligo (dT) primer containing T7 promoter sequence, converted into double-stranded cDNA, and used for generation of Cy3-labelled complementary RNA (cRNA) by T7 RNA polymerase.The Cy3-labelled cRNAs were cleaned using Qiagen RNeasy columns (Qiagen, Germany, Cat No. 74106).1650 ng of Cy3-labelled cRNA was hybridized on an Agilent wheat 4x44K microarray (AMADID 22297, Agilent Technologies, USA) using Agilent Gene Expression Hybridization Kit (Part No. 5190-0404), and inside Agilent Sure hybridization chamber for 16 hours at 65˚C.Microarray slides were washed using Agilent Gene Expression wash buffer (Part No. 5188-5327) and scanned on G2600D microarray scanner (Agilent technologies, USA).Extraction of data from microarray slide images was done using Agilent Feature Extraction software Ver-11.5 and analysed by Agilent GeneSpring GX Version 12 (GS) software.Briefly, the signals were corrected for background and baseline transformed to the median of all spots, intra-and inter-microarray normalization was done for all the samples.Principal Component Analysis (PCA) was done to assess quality of independent replicates, and global normalization (normalized by 75 th percentile shift method) of spot intensities was done using GS software (S1 Fig).Spot intensities were log 2 transformed and averaged for two replicate spots.DEGs were identified using unpaired student t-test having a Fold Change (FC) value � 2.0 with Benjamini-Hochberg FDR corrected p-value � 0.05, and visualized in volcano plots (S2 Fig) [32].Expression profiles of DEGs were analyzed by hierarchical clustering complete linkage method using Heatmap Illustrator Ver 2.0 [33].The microarray data files have been submitted to the Gene Expression Omnibus (GEO) database (accession no GSE207175).

Functional annotation of DEGs
Transcript consensus (TC) sequences specific to the microarray probes were retrieved from Triticum aestivum gene indices TAGI 12 release of JCVI/TIGR plant gene indices [34].TC sequences were used for similarity search analysis against the wheat RefSeq v2.0 (genotype: Chinese Spring) standard and used for further analysis [35].Biological functions were assigned using Gene Ontology (GO) annotation for Molecular Function (MF), Biological Process (BP) and Cellular Component (CC) using quickGO [36].Enrichment and visualization of GO terms was done using ShinyGO tool [37].Important pathways affected by these DEGs were identified using KEGG KOLA and DAVID database [38][39][40].

Validation of differential gene expression by RT-qPCR analysis
10μg of isolated total RNA was reverse transcribed using Superscript III Reverse transcriptase (Life Technologies, USA) and Oligo (dT) 20 primer (Life Technologies, USA), as per the recommended protocol.Gene-specific primers were designed using Primer3 software and evaluated for specificity with NCBI Primer BLAST (S1 Table) [41].Real time PCR assays were carried out on Eppendorf realplex 4 thermal cycler (Eppendorf, Germany) using Sigma SYBR Green JumpStart Taq Ready Mix (Sigma Aldrich, USA) as per manufacturer's instruction.Data analysis was done using Eppendorf realplex software Ver2.2.Wheat Actin gene (TaAct) was used as reference gene for normalizing the expression of each gene and FC was calculated using 2 -ΔΔCt method [42].The RT-qPCR analysis was repeated three times with independent biological replicates.

Mapping of stem rust responsive DEGs to the Thinopyrum elongatum genome
The stem rust responsive DEGs were mapped to the T. elongatum chromosomal region harbouring the Sr24-linked marker using the following approach.Chromosome 3E of T. elongatum genomic sequence (NCBI genomes reference genome number ASM1179987v1), was used for mapping of Sr24 tightly linked SSR marker Xbarc71 (NCBI GenBank id BV211796.1)and stem rust responsive DEGs using NCBI-BLAST tool.The mapped DEGs were filtered to 70Mbp region from end terminal of 3E chromosome (equivalent to approximately ±2 cM from Xbarc71 SSR marker).The mapped gene coordinates were visualized using ChromoMap Ver 4.1.1[43].

Wheat NILs phenotypic reaction to stem rust pathogen
The wheat NILs (C306, susceptible and HW2004, resistant) differing in Sr24 stem rust resistance gene showed differential rust reaction post inoculation of the pathogen, Pgt pathotype 7G11.Inoculated plants started showing visible response to stem rust infection 5 dpi, where HW2004 showed reduced yellow flecks compared to C306 (Fig 1A and 1B).At 9 dpi brick red coloured fungal spore pustules or uredospores started to appear in flecked regions in C306, whereas the HW2004 showed minute uredospores surrounded by large flecks, indicative of hypersensitive immune response against the pathogen (Fig 1C).At 14 dpi, uredospores showed profuse growth on the leaf surface of C306, compared to HW2004, where it was largely restricted (Fig 1D).Appearance of HR symptoms are typical of resistance response to fungal pathogen in plants, and have also been reported in wheat leaf as well as stripe rust infections [16,19].

Wheat NILs exhibit transcriptomic differences in response to stem rust
The current study employed NILs for stem rust resistance gene Sr24, developed after seven backcrosses thus minimizing the background transcriptomic differences [44].Previous studies on wheat leaf and stripe rust have reported activation of various defence response within few hours of pathogen inoculation [17,18].In the current study the transcriptomic profiles were studied at basal level (0 hpi), and to capture early as well as late events after infection (early: 10 hpi and late: 72 hpi), and DEGs at these stages were identified (S3 Fig, S2-S4 Tables).Expression profiles showed higher number of DEGs in HW2004 (894 genes, including higher number of upregulated DEGs) compared to C306 (665 genes) indicating differential transcriptomic reprogramming upon Pgt infection ( In HW2004 at early stage (10 hpi), 612 genes were found to be upregulated (524 uniquely at early, 88 at both early and late stage) and 546 were downregulated (500 only at early, 46 at both early and late stage).At late infection stage (72 hpi), the number of DEGs decreased significantly, with 282 upregulated (194 unique at late stage) and 395 downregulated (349 unique at late stage) genes (Fig 3A -3C).In case of C306, response at early infection stage was characterized by, upregulation of 416 genes (339 unique at early, 77 at early and late stages), which was relatively lower than HW2004, while downregulation was observed for 615 (600 uniquely at early, 15 at early and late stages) genes.At late stage (72 hpi) the number of DEGs decreased with 249 upregulated and 426 downregulated (Fig 3D -3F).
High genetic similarity between the NILs C306 and HW2004 was evident in the minor transcriptomic differences at basal levels, with only 29 genes with higher and seven with lower transcript levels in HW2004 (  1 and 2).

HW2004 showed early pathogen detection and hypersensitive response
Early stage.In HW2004 peaking of transcriptomic response at early stage suggests that pathogen recognition and activation of defence response is an early event in wheat Pgt interaction.Among the upregulated DEGs important candidates included pathogen receptors and defence related TFs viz.NBS-LRR, Myb-like DNA-binding domain containing proteins.Genes involved in HR and neutralizing pathogen including SGNH hydrolase-type esterase, cytochrome P450, sugar efflux transporter, dirigent protein, phosphomethylpyrimidine synthase, Caffeate O-methyltransferase (COMT), 2OG-Fe(II) oxygenase, lipoxygenase were also upregulated (Table 1, Figs 5, 6A-6F, S2 Table ).Further gene-based clustering and pathway analysis showed, that the upregulated genes were associated with signalling, response to stress, transporter activity, TF binding activity, cytoskeletal reorganization, HR mediating The upregulation of signalling cascade at early stages of infection have also been reported in previous studies in case of wheat Lr10 gene response, as well as other plant diseases [45][46][47][48].Similar to current findings, Lr28-mediated resistance response has also shown enhanced cytoskeletal reorganization upon rust infection [49][50][51].The HW2004 showed downregulation of genes related to chlorophyll a/b-binding protein, multiple Myb type TFs and zinc finger (B-Box type) containing protein (Table 1, S2 Table ).In, addition some signalling genes (kinases, signal transduction) and TFs were also downregulated, suggesting that these might not be involved in defence response to Pgt in wheat (Figs 5 and 6).
Late stage.At late infection stage (72 hpi), the number of DEGs decreased significantly (Fig 3A -3C).The upregulated genes that showed sustained defence response, included genes for BTB domain containing protein, nudix hydrolase, glycosyl hydrolases, cytochrome b6-f complex subunit 5, defensin, nucleases and senescence domain containing protein (Table 2, S2 Table ).GO annotation showed these genes to be related to signalling cascade, defence mechanism (NBS-LRR and ABC transporter protein), TFs, lipid metabolic, ROS burst, calcium flux modulating genes (EF hand calcium-binding domain profile protein), along with several unannotated genes ( At late infection stage downregulated genes included dirigent protein, agglutinin domain containing protein, germin-like protein, photosystem II related protein, reported to be downregulated in other rust diseases (Table 3, S2 Table) [50,51].These genes are primarily associated with basic growth and physiological processes and metabolic pathways (protein,

C306 showed impaired pathogen detection and defence response
Early stage.In case of susceptible genotype C306, response at early infection stage (10 hpi) was characterized by, upregulation of relatively lower number of genes than HW2004 (Fig 3D -3F).Lower number of upregulated genes in susceptible reaction, has been previously associated with faster growth and early establishment of pathogen [19,52].The downregulated genes included multiple hydrolases, Myb-type TFs involved in defence, proteins involved in detoxification (MATE efflux family protein), and candidates involved in pathogen sensing (serine/threonine kinases, NBS-LRR domain containing protein, serine/threonine protein kinases, major facilitator of sugar superfamily protein, MFS), suggesting an impaired pathogen detection, and absence of pathogen-specific defence responses (S3 Table ).Although C306 also showed upregulation of some genes involved in signalling, transporters and ROS modulation,   ).As, these genes are associated with important defence related GO categories viz.response to biotic/abiotic stimuli and associated with downstream signalling, their downregulation is indicative of sustained failure of defence response (Fig 5, S3 Table ).The upregulated genes consisted of few candidates central to plasma membrane localized stress responsive functions viz.signal receptor binding, kinases and transporters, but relatively lower in numbers and expression levels, indicating inadequate defence response to restrict pathogen growth.However, in contrast to HW2004, several metabolic, biosynthetic process related genes were It is very likely that some of these highly upregulated genes are responsible for susceptibility, by suppressing defence related genes [50,51].

Differential transcriptomic response in HW2004 and C306 upon Pgt infection
Wheat NILs showed DEGs at basal level.The two NILs showed minor differences at basal levels (Fig 4A).In the HW2004, basally, upregulated candidates included, GMC oxidoreductase, Hsp20, MFS type sugar transporter, SKP-1 like protein and an NBS-LRR, various Rproteins, transketolases and CED-4 domain containing protein (S4 Table ).The NB-ARC domain and sugar transporter protein belong to 'R' gene family and involved in resistance to leaf and stripe rusts [5].In addition, transketolase, oxidoreductase, and Hsp proteins are identified as key components of biotic stress response [53,54].Higher basal level of candidates, particularly those involved in pathogen detection, downstream signalling, defence mechanisms suggests enhanced levels of defence preparedness before pathogen infection, which may be central to "R" gene mediated resistance.Such basal expression differences of defence related genes has also been reported to contribute towards resistance to Xanthomonas infection in rice [48,55].Present study identified several unannotated genes with substantially higher basal levels in HW2004, viz.TC453923 (FC 8.33), TC443516 (FC 6.83), TC409011 (FC 6.59).These genes may serve as important candidates for further characterization to decipher their roles in stem rust resistance in wheat.The major downregulated DEGs included nuclear pre-mRNA splicing factor, 40S ribosomal protein, pectin acetyl esterase and some unknown genes.Few genes related to broad spectrum resistance viz.chitinase, peroxidase, displayed lower levels, indicating that these may not be involved at basal level and might be required at later stages (S4 Table ).
HW2004 detects pathogen and activate HR at early stage.Comparative analysis at early stage of infection (10 hpi) revealed upregulation of candidates involved in pathogen recognition, defence signalling, TFs involved in activation of defence responsive genes in HW2004 (Fig 6, Table 5).Activation of HR, ABC transporters, PR proteins, glucosidases, fungal cell wall dissolving enzymes (pectin-glucuronyl transferase, glucanase, pectinase, hydrolases), ROS generation, and lipid metabolism genes was also observed (Fig 5, S4 Table ).Similar involvement of early stage genes have been reported in wheat leaf and stripe rust response, in detection of effectors and activation of defence response [19,50].Early HR induction in presence of 'R' gene is a hallmark of specific defence response in plants [8].The genes showing lower levels included a jasmonate induced protein, and multiple DEAD/DEAH box helicases, nuclear presplicing factor, and molecular chaperone SugE (Table 5, S4 Table ).In general, pathways related to primary metabolism (carbohydrates, energy, amino acids, and lipids), and cellular process (processes related to cell growth, death and cellular community) were also downregulated (S7 Fig) .Reduced but sustained defence response at late stages in HW2004.At 72 hpi number of DEGs were significantly reduced (Fig 4A -4C), which suggests that the modulation of crucial transcriptomic responses against stem rust pathogen occurs primarily at the early stages of infection.Findings in rice have also shown an early transcriptomic peaking response upon pathogen attack followed by a coordinated modulation of gene expression at later stages [18,48].Important upregulated genes included transket_pyr, BTB and Skp1 domain-containing protein, Mob1/phocein family protein (S4 Table ).The upregulated genes were found to affect pathways related to genetic and environmental information processing, metabolism of lipids, terpenoids and polyketides, along with few involved in carbohydrate and protein metabolic pathways (S7 Fig) .DEGs modulated at late stages of infection (2 to 5 dpi) are postulated to be associated with defence related pathways including HR, phytohormones mediated defence pathways, cell wall fortification mechanism, lipid peroxidation and modulation of carbohydrate metabolism [18,47].Also, levels of some of the immediate stress responsive GO groups viz.response to toxic substance, anchoring junction, ROS burst (oxidative stress response, H 2 O 2 catabolism, and detoxification) were relatively reduced, indicative of their importance in the early infection stage (Figs 5 and 6).
Expression profile by RT-qPCR of representative genes from different categories (defence, hypersensitive response, primary and secondary metabolism, unannotated) showed that their trend was consistent with microarray data (S8 Fig) .RT-qPCR analysis of important candidates was carried out in both the NILs (Fig 7A and 7B), The HW2004 displayed upregulation of key signalling genes (kinases and NBS-LRR domain containing proteins) and TFs (WRKY45) involved in activation of defence responses (at early and late stages), while levels of these were lower in C306.Further, HW2004 also showed upregulation of genes associated with hypersensitive response (PR proteins, β-1,3-glucanase, Cytochrome P450), secondary metabolites (Caffeic acid O-methyltransferase, Chalcone synthase) and ROS modulation (peroxidases) compared to C306 that showed relative lower levels these genes, except those involved in some secondary metabolite synthesis and ROS modulation were upregulated, but only at later stages (Fig 7A and 7B).
Important components of resistance against stem rust of wheat.Differential expression analysis of wheat NILs upon Pgt infection, identified several key aspects mediating stem rust resistance response, including pathogen detection (signal receptor and transducing), activation of defence TFs and downstream induction of multiple defence processes, collectively helping in restricting pathogen growth.

Signalling
Pathogen detection and signalling genes are crucial for early detection of infection and activation of defence responses via a complex cascade of pathways [11].Upregulation of key signalling genes viz.Ser/Thr kinases, MAP Kinases and NBS-LRR specifically in HW2004, might be important for activation of HR and other defence mechanism against stem rust.HW2004 showed upregulation of higher number of NBS-LRR genes compared to C306, a known 'R' signature (Fig 6, Tables 1 and 2, S2 Table ).Other studies have also shown similar expression profiles (both upregulated and downregulated) of NBS-LRR genes upon pathogen infection [50,51].Additionally, calcium and phosphatidylinositol signalling systems were also upregulated in the HW2004, which was consistent with a similar regulatory trend reported in case of Lr28 [50,51].Likewise, cyclin-dependent protein kinases, cysteine rich receptor like protein kinases, diacylglycerol kinase 5, have also been reported to be important in other wheat rust interaction studies [16,47,48,56,57].

HR pathways
Biotic stress induced TFs are involved in activation of defence pathways.Several key genes central to the HR during plant pathogen interaction (ABC transporters, Cytochrome P450, WIR1, Caffeic acid-O methyltransferase (COMT), heat shock proteins, chitinase, alcohol dehydrogenase, ankyrin repeat profile containing protein) were upregulated in the HW2004, in response to stem rust infection (Fig 6, Tables 1 and 2, S2 Table ).Moreover, HW2004 also displayed upregulation of candidates involved in HR signalling (phosphoinositide specific phospholipase), biosynthesis of secondary metabolites, SA biosynthesis and Systemic Acquired Resistance (SAR) (PR1, PR4, defensin, isochorismate synthase, UDP-glucosyltransferase, methylesterase) [2,6].Expression analysis also indicate cross talk between basal-and race-specific defence responses, as seen in other wheat rust interaction studies [50].

Transporters
HW2004 showed upregulation of multiple transporter proteins (ABC, MFS type, zinc/iron) and many proteins with transporter domain (Fig 6, Tables 1 and 2, S2 Table ).Such changes can potentially alter the intracellular metabolite conditions and may contribute to resistance against the pathogen, as shown in case of sugar transporters and other transmembrane transporters [5,58].Resistance mechanism in Lr34 and Lr10 have showed upregulation of ABC, MATE efflux, pleiotropic drug transporters [16,47], while upregulation of ion flux transporters (Ca 2+ , K + , H + ) have been reported in resistance response to Xanthomonas in rice [48].

ROS modulation
Defence mechanisms activate ROS generation to restrict the pathogens [5,11], and genes involved in ROS modulation are involved in the response to fungal pathogen infection in wheat [16,47,48,50,51].In the current study, significant upregulation of peroxidases (ascorbate, glutathione), catalases, thioredoxins, multiple superoxide dismutase, glutathione-S-transferase, alternative oxidases were observed in HW2004 (Fig 6, Tables 1 and 2, S2 Table ).Enhanced ROS levels, during initial stages of infection (starting from initial haustoria formation) helps in restricting the pathogen growth, however, at later stages a dynamic balance of ROS generation and elimination is maintained by the host to efficiently encounter pathogen along with maintenance of its redox environment [52].

Secondary metabolites
Secondary metabolites such as flavonoids (derived from phenylpropanoid metabolism) comprise an integral component of plant defence responses against pathogens [56].Genes involved in secondary metabolite biosynthesis and xenobiotic biodegradation were consistently upregulated in HW2004 (Fig 6, Tables 1 and 2, S2 Table ).The phenylpropanoid pathway is responsible for biosynthesis of antipathogen compounds (anthocyanins, lignin, and phytoalexins), and is known to be induced for rust resistance mediated by Lr34 gene [16].Phenylalanine ammonia lyase (PAL, involved in synthesis of SA) also mediates biosynthesis of lignin leading to lignification and strengthening of cell wall against pathogen invasion [52].Lipoxygenases a key enzymes of lipid metabolism is also involved in Jasmonic acid (JA) based signalling for defence response [48].

Metabolic pathways
Defence response to pathogen also involves modulation of primary metabolic pathways (catering for energy requirement), to restrict pathogen growth.Genes involved in steroid biosynthesis and, linoleic acid metabolism were upregulated in HW2004 (Fig 6, Tables 1 and 2, S2 Table ).This was consistent with the previously reported defence response to Xanthomonas [48].Overall, carbohydrate metabolism was also modulated in both resistant and susceptible NILs.HW2004 showed downregulated tricarboxylic acid (TCA) cycle and upregulated glycolysis, suggesting preference for faster energy production, also use of alternate carbohydrate source (galactose).Previous report on Lr34 response have also shown modulation of TCA cycle and GABA shunt pathway, while, Lr1 response study showed upregulation of glycolysis [16,18].

Uncharacterized genes
Several uncharacterized genes were substantially upregulated in the resistant wheat NIL HW2004, some of which are likely to be involved in resistance to stem rust, with possible roles in detection of pathogen, or defence responses (Fig 6, Tables 1 and 2, S2 Table ).Further characterization of these candidate genes will provide a better insight into molecular basis of wheat stem rust interaction.containing proteins, and TFs, suggesting their role in defence activation.In addition, some DEGs belonged to defence response categories viz.HR, Transporters and Hydrolases.These are potential candidates for primary transcriptional signal response upon Pgt infection, which further activates an elaborated host defence response.Among the downregulated DEGs 16 were from early stage and seven from later stage of infection which were primarily related to metabolic and photosynthetic roles (S5 Table ).It is interesting to note that many of the uncharacterized/unannotated DEGs also mapped to T. elongatum translocated fragment and were close to the Sr24-linked SSR marker Xbarc71 (Fig 8 ), some of which might be important for the rust resistance.The limited understanding of roles of the genes originating from these wild relatives of wheat, advocates their detailed characterization for exploitation of their hidden potential in disease resistance breeding [24].

Conclusion
Current study utilized wheat NILs for stem rust resistance gene Sr24, for studying transcriptomics difference in response to Pgt race 7G11.Comparative transcriptomic analysis revealed higher basal levels of genes involved in defence to pathogen, in resistant NIL HW2004.HW2004 also showed early pathogen detection and defence responses with activation of plasma membrane associated receptors coupled with kinases, suggesting initiation of complex signalling cascades to activate both broad spectrum and specific defence responses.Study suggests that a combination of specific and basal defence response involving ROS generation, cell wall fortification, PR proteins, antifungal products of phenylpropanoid pathways and hydrolases, seems to be responsible for restricting the pathogen growth (Fig 9).Further, many highly over and under expressed uncharacterized genes were identified.These genes may be important in defence response to stem rust and may be characterized to decipher molecular nature and significance in future.Overall this study is helpful in understanding the molecular basis of defence response to stem rust in wheat.

Fig 2 .
Fig 2. Global expression profile of DEGs in response to Pgt infection.Overview of transcriptomic response at three time-points (0 hpi, 10 hpi and 72 hpi) in (A) HW2004 and (B) C306 based on hierarchal clustering.Up refers to upregulated and Down to downregulated DEGs in the respective NILs, R1 & R2 refer to the two independent biological replicates.https://doi.org/10.1371/journal.pone.0295202.g002

Fig 3 .
Fig 3. Number of DEGs at early (10 hpi) and late (72 hpi) stages of infection in the two NILS.(A) Number of DEGs at 10 hpi and 72 hpi compared to 0 hpi time-point in HW2004.(B) Venn diagram representation of common and unique upregulated DEGs at 10 hpi and 72 hpi in HW2004.(C) Common and unique downregulated DEGs at 10 hpi and 72 hpi in HW2004.(D) Number of DEGs at 10 hpi and 72 hpi compared to 0 hpi time-point in C306.(E) Venn diagram representation of common and unique upregulated DEGs at 10 hpi and 72 hpi in C306.(F) Common and unique downregulated DEGs at 10 hpi and 72 hpi in C306.Numbers in the Venn diagram represent number of DEGs in the respective stage of infection.https://doi.org/10.1371/journal.pone.0295202.g003

Fig 4 .
Fig 4. Comparative transcriptomics of wheat NILs for Sr24.(A) Number of DEGs in HW2004 compared to C306 at three stages of infection (0, 10 and 72 hpi).Venn diagram representation of number of unique and shared DEGs at different stages of infection: (B) Upregulated and (C) Downregulated genes in HW2004 at basal, early and late stages of infection.Common and unique DEGs among the two NILs at early (10 hpi) stage of infection: (D) upregulated and (E) downregulated genes.Numbers in the Venn diagram represent number of DEGs in the respective category.https://doi.org/10.1371/journal.pone.0295202.g004

Fig 5 .Fig 6 .
Fig 5. Gene Ontology (GO) characterization of upregulated genes.Overview of GO terms in (A) HW2004 and (B) C306.BP refers to Biological Process, MF to Molecular Function and CC to Cellular Compartment.The GO IDs of the respective GO terms are indicated in the parenthesis.https://doi.org/10.1371/journal.pone.0295202.g005

Fig 8 .
Fig 8. Characteristics of the DEGs in Wheat NIL HW2004 (post Pgt 7G11 infection) mapped on the 70 Mbp region of the translocated fragment (spanning the Sr24-linked marker, Xbarc71) corresponding to the chromosome 3E of Thinopyrum elongatum.A) Relative positions of Sr24-linked marker Xbarc71 and DEGs mapped across the 70 Mbp region of the translocated fragment.B) Relative expression levels (log 2 FC) of the DEGs.C) Biological functional categorization based on the Uniprot/TC ID information.D) Indication of post-infection time-course expression response (early/late and up/down) of the mapped DEGs.The scale on the bottom is indicative of the position of the DEGs in the 70 Mbp region, while the information about the functional categories (and colour codes) are indicated on the right-hand side panel.https://doi.org/10.1371/journal.pone.0295202.g008

Fig 9 .
Fig 9. Schematic representation of probable mechanism of Sr24-mediated resistance in wheat upon Pgt infection.Black coloured lines represent sequence of events and candidates involved in pathogen detection and activation of defence responses.Red coloured lines and shapes represent upregulated pathways and processes upon Pgt infection, while blue coloured lines represent pathways which are repressed upon Pgt infection.https://doi.org/10.1371/journal.pone.0295202.g009

Table 5 . Functional annotation of representative important differentially expressed genes in HW2004 compared to C306 at early infection stage.
$ Based on Uniprot annotation; N/A Gene ID not retrievable https://doi.org/10.1371/journal.pone.0295202.t005